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View Code? Open in Web Editor NEWOlivar: automated variant aware primer design for multiplex tiled amplicon sequencing
Home Page: https://doi.org/10.1101/2023.02.11.528155
License: MIT License
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liushuailongolivar's Issues
Limit BLAST search with taxid
Use the get_species_taxids.sh script (comes with blast) to get all taxids below a non-leaf taxid.
Ambiguous bases in primer design?
Hi @treangen
Olivar is very interesting in it's approach to primer design. I'm testing it out for a few different organisms. I noticed that while reference can not contain an ambiguous bases, the variation file can have ambiguous bases. However, even if they are included in the variation file, it seems Olivar ignore them in the primers. Interestingly, the graphical output does include the SNP.
It would be super helpful if when Olivar decides on a primer in a particular location where an ambig base exists, the primer would also contain such a base.
Thanks!
Ammar
Error message for variant position out of index
Support for multiple genomes input
Shouldn't be hard, but how to format the risk array figure? Output multiple figures?
The --*-prefix flags cause issues
Hi,
Olivar is a great tool, thanks for creating and maintaining it. I'm using it to design primers for Hep A. This is the command I am running:
olivar tiling \
output/4.olivar_reference/olivar-ref.olvr \
-o output/5.olivar_primers \
--max-amp-len 1200 \
--min-amp-len 1000 \
--seed 1995 \
--fp-prefix HAVSeq \
--rp-prefix HAVSeq \
--check-var
It produces this error:
Successfully loaded reference file output/4.olivar_reference/olivar-ref.olvr
reference sequence length: 7478
design region: 16:7463
region length: 7448
number of PDR sets to be tested: 3115
Designing PDRs...
100%|>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>| 3115/3115 [01:19<00:00, 38.98it/s]
Finished in 79.921s
Best total risk: 1151.410
total amplicons: 10
covered region: 90:7217
coverage of reference sequence: 95.320%
Generating primer candidates...
0%| | 0/10 [00:00<?, ?it/s]ta
Traceback (most recent call last):
File "micromamba/bin/olivar", line 156, in <module>
tiling(
File "micromamba/lib/python3.10/site-packages/olivar/main.py", line 793, in tiling
all_plex_info_primer = get_primer(all_plex_info, config)
File "micromamba/lib/python3.10/site-packages/olivar/main.py", line 436, in get_primer
fP, fail = fP_generator.get(fP_design, fP_prefix, check_BLAST=check_BLAST, **fP_setting)
File "micromamba/lib/python3.10/site-packages/olivar/design.py", line 226, in get
primer_arr.append(basic.seq2arr(s))
File "micromamba/lib/python3.10/site-packages/olivar/basic.py", line 76, in seq2arr
seq_list = [dic[s] for s in list(seq_str)]
File "micromamba/lib/python3.10/site-packages/olivar/basic.py", line 76, in <listcomp>
seq_list = [dic[s] for s in list(seq_str)]
KeyError: 'H'
I modified the function seq2arr causing the error to print the output seq_str , this was the print out:
ct
gc
gg
ag
ta
ct
cc
gc
tg
tt
tt
gt
cg
cc
gc
cg
HAVSeqcgccgtttgcctaggcta
Note the last sequence has HAVSeq inserted. Removing the prefix flags solves the issue. It seems that somehow the primer names are being inserted into the sequence operation. I have tested the following:
- including both prefix flags (error)
- including only one prefix flag (error)
- quoting the input eg
--rp-prefix "HAVSeq"(error) - not including any prefix flag - works!
I am running on Linux Mint 21, with micromamba installing Olivar version 1.1.1. Also tested with the latest version 1.1.2, same issue.
Not a huge deal as the Olivar still runs, just need to manually rename the outputs.
Optimization for short input genome
When input genome is short compared to desired amplicon length (e.g., less than 20 amplicons to cover the whole genome), optimize for genome coverage. Loss should be multiplied by 1/coverage.
Validate module errors out with "KeyError: 'r'"
Hello,
I am trying to validate a primer scheme and I get a keyerror message, see below:
(olivar) cygnus@pop-os:/media/extra_drive1/XXXX_tiled_amplicon_assay/olivar_v1.1.3_XXXX_scheme_development/XXXX_olivar_nonspecific_genomes$ olivar validate /media/extra_drive1/XXXX_tiled_amplicon_assay/testing_XXXX_denv_primers/DENV4_primers.csv --pool 1 -d /media/extra_drive1/XXXX_tiled_amplicon_assay/olivar_v1.1.3_XXXX_scheme_development/XXXX_olivar_nonspecific_genomes/XXXX_olivar_nonspecific_db.fa -o /media/extra_drive1/XXXX_tiled_amplicon_assay/testing_XXXX_denv_primers/olivar_validate/denv4_pool1 -p 20
Successfully loaded /media/extra_drive1/XXXX_tiled_amplicon_assay/testing_XXXX_denv_primers/DENV4_primers.csv, pool-1.
Running blastn-short with 20 CPU(s), batch size 128...
100%|>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>| 36/36 [00:54<00:00, 1.51s/it]
finished in 54.47s
Non-specific amplicons saved as /media/extra_drive1/XXXX_tiled_amplicon_assay/testing_XXXX_denv_primers/olivar_validate/denv4_pool1/olivar-val_pool-1_ns-amp.csv
Non-specific primer pairs saved as /media/extra_drive1/XXXX_tiled_amplicon_assay/testing_XXXX_denv_primers/olivar_validate/denv4_pool1/olivar-val_pool-1_ns-pair.csv
Traceback (most recent call last):
File "/home/cygnus/mambaforge/envs/olivar/bin/olivar", line 187, in <module>
validate(
File "/home/cygnus/mambaforge/envs/olivar/lib/python3.12/site-packages/olivar/main.py", line 1150, in validate
_, all_bad = PrimerSetBadnessFast(seq_list)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/cygnus/mambaforge/envs/olivar/lib/python3.12/site-packages/olivar/design.py", line 310, in PrimerSetBadnessFast
c = basic.revcomp(p)
^^^^^^^^^^^^^^^^
File "/home/cygnus/mambaforge/envs/olivar/lib/python3.12/site-packages/olivar/basic.py", line 36, in revcomp
bases = [complement[base] for base in seq]
~~~~~~~~~~^^^^^^
KeyError: 'r'
The output directory is created and the "olivar-val_pool-1_ns-amp.csv" and "olivar-val_pool-1_ns-pair.csv" are created but are empty (just have headers). Any help would be appreciated. Thanks!
-Alex
Add container images for different OS (including Windows)
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